LOCUS       sequence-337814-        5517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription for rescue experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5517)
  AUTHORS   Lee YR, Kim SH, Ben-Mahmoud A, Kim OH, Choi TI, Lee KH, Ku B, Eum J,
            Kee Y, Lee S, Cha J, Won D, Lee ST, Choi JR, Lee JS, Kim HD, Kim HG,
            Bonkowsky JL, Kang HC, Kim CH
  TITLE     Eif2b3 mutants recapitulate phenotypes of vanishing white matter 
            disease and validate novel disease alleles in zebrafish.
  JOURNAL   Hum Mol Genet. 2021 Apr 27;30(5):331-342. doi: 10.1093/hmg/ddab033.
  PUBMED    33517449
REFERENCE   2  (bases 1 to 5517)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Hum Mol Genet"; date: "2021-04-27- 27"; volume: "30"; issue: "5"; 
            pages: "331-342"
COMMENT     Sequence Label: EIF2B3-V30A
FEATURES             Location/Qualifiers
     source          1..5517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1355..1366
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     polyA_signal    2547..2681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2571..2590)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2625..2644
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2794..2814)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2794..2812)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2833..2849)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2833..2849)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2846..2868)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2857..2873
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2881..2887,2888..2905,2906..2911))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2926..2947
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3064..3081)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3235..3823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3315..3334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3994..4785,4786..4854))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4617..4636
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4855..4959)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4985..5440)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5122..5143)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5334..5353
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat cccatcgatg gccgcacaca
     1081 agctggctga cccggtttgt aaaaatggaa tttcaagcag tagtgatggc agtaggtgga
     1141 ggatctcgga tgacagacct aacttccagc attcccaaac ctctgcttcc agctgggaac
     1201 aaacctttaa tttggtaccc attgaacctg cttgagcgtg ttggatttga agaagtcatt
     1261 gtggttacaa ccagggatgt tcaaaaggct ctatgtgcag aattcaagat gaaaatgaag
     1321 ccagatattg tgtgtattcc tgatgatgct gacatgggaa ctgcagattc tttgcgctac
     1381 atatatccaa aacttaagac agatgtgctg gtgctgagct gtgatctgat aacagacgtt
     1441 gccttacatg aggttgtgga cctgtttaga gcttatgatg catcacttgc tatgttgatg
     1501 agaaaaggcc aagatagcat agaacctgtt cccggtcaaa aggggaaaaa aaaagcagtg
     1561 gagcagcgtg acttcattgg agtggacagc acaggaaaga ggctgctctt catggctaat
     1621 gaagcagact tggatgaaga gctggtcatt aagggatcca tcctacagaa gcatcctaga
     1681 atacgtttcc acacgggtct tgtggatgcc cacctctact gtttgaaaaa atacatcgtg
     1741 gacttcctaa tggaaaatgg gtcaataact tctatccgga gtgaactgat tccatattta
     1801 gtgagaaaac agttttcctc agcttcctca caacagggac aagaagaaaa agaggaggat
     1861 ctaaagaaaa aggagctgaa gtccttagat atctacagtt ttataaaaga agccaataca
     1921 ctgaacctgg ctccctatga tgcctgctgg aatgcctgtc gaggagacag gtgggaagac
     1981 ttgtccagat cacaggtgcg ctgctatgtc cacatcatga aagaggggct ctgctctcga
     2041 gtgagcacac tgggactcta catggaagca aacagacagg tgcccaaatt gctgtctgct
     2101 ctctgtccag aagaaccacc agtccattcg tcagcccaga ttgtcagcaa acacctggtt
     2161 ggagttgaca gcctcattgg gccagagaca cagattggag agaagtcatc cattaagcgc
     2221 tcagtcattg gctcatcctg tctcataaaa gatagagtga ctattaccaa ttgccttctc
     2281 atgaactcag tcactgtgga ggaaggaagc aatatccaag gcagtgtcat ctgcaacaat
     2341 gctgtgatcg agaagggtgc agacatcaag gactgcttga ttggaagtgg ccagaggatt
     2401 gaagccaaag ctaaacgagt gaatgaggtg atcgtgggga atgaccagct catggagatc
     2461 tgagttctga gcaagtcaga ctccttcctt ttggcctcca aagccacaga tgttggctct
     2521 agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg
     2581 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2641 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     2701 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc
     2761 caatgcattg ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     2821 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2881 caacatacga gccgggagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     2941 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3001 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3061 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3121 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3181 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3241 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3301 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3361 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3421 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3481 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3541 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3601 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3661 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3721 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3781 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3841 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3901 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3961 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4021 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4081 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4141 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4201 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4261 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4321 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4381 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4441 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4501 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4561 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4621 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4681 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4741 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4801 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4861 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4921 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4981 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5041 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5101 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5161 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5221 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5281 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5341 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5401 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     5461 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgacc
//
